Biosynthesis of a fluorescent protein with extreme pseudo-Stokes shift by introducing a genetically encoded non-natural amino acid outside the fluorophore

A novel kind of fluorescent protein relying on the intramolecular interplay between two different fluorophores, one of chemical origin and one of biological origin, was developed. The fluorescent non-natural amino acid l-(7-hydroxycoumarin-4-yl)ethylglycine was site-specifically incorporated into the recombinant enhanced cyan fluorescent protein (eCFP) at a permissible surface position ～20 ? away from the protein fluorophore using amber suppression in Escherichia coli with an engineered cognate Methanococcus jannaschii tRNA synthetase. The resulting eCFP(Cou) exhibited almost quantitative intramolecular Fo?rster resonance energy transfer (FRET) between its two fluorophores, showing brilliant cyan emission at 476 nm upon excitation in the near-UV at 365 nm (a wavelength easily accessible via conventional laboratory UV sources), in contrast to its natural counterpart. Thus, this fluorescent protein with unprecedented spectroscopic properties reveals an extreme apparent Stokes shift of ～110 nm between the absorption wavelength of the coumaryl group and the emission wavelength of eCFP.     

Quantitative determination of chloride by means of flow injection analysis with spectrophotometric detection in the UV/VIS range

Chloride is determined indirectly by Spectrophotometric flow injection analysis. Two systems are compared, both based on the principle of ion exchange of easily detectable anions versus chloride from suitable mercury salts. The first method is based on the exchange of chloride with chloranilate which is detected at 332 nm or at 306 nm in neutral or in acidic medium respectively. In the second case, chloride reacts with Hg(SCN)₂. The liberated thiocyanate forms a strongly coloured complex with Fe(III) in acidic solution with an absorption maximum at 460 nm. Both methods have a detection limit of about 5 mol Cl^–/l (175 ng/ml). In the case of the thiocyanate method, the relative standard deviation is about 2% (7 measurements) in the range of 5 to 150 mol/l and decreases significantly to a value of approximately 0.2% at higher concentrations; for the chloranilate method it is 10% for lower and about 1% for higher concentrations respectively.     

Mass spectrometric fragmentation of isomeric 2-alkyl-substituted 1,3-indandiones and 3-alkylidenephthalides: a seven-step consecutive isomerization of regular and distonic molecular radical cations

The electron impact-induced fragmentation of 2,2-dimethyl- and 2-ethyl-1,3-indandione, 1 and 2, and their isomers, 3-isopropylidene- and 3-propylidenephthalide, 3 and 4, respectively, was studied in detail by mass-analysed ion kinetic energy (MIKE) and collision-induced dissociation (CID-MIKE) spectrometry, including ²H and ¹³C. labelled analogues of 1 and 2. In all regimes of internal energy, the molecular ions 1^+. ? 4^+. interconvert by up to seven consecutive, reversible isomerization steps prior to the main fragmentation processes, viz. loss of CH₃^. and C₂H₄. 1,3-Indandione and 3-methylenephthalide ions with identical alkylidene moieties (i.e. 1^+.?3^+. and 2^+.?4^+.) equilibrate rapidly and completely prior to fragmentation, whereas these pairs of isomers interconvert only slowly via a five-step rearrangement of the indandione ions 1^+.?2^+.. Distinct from the behaviour of simpler ionized carbonyl species, a 1,2-C shift of a (formally) neutral carbonyl group is found to occur along with that of a protonated one. Also distinct from simpler cases, methyl loss does not take place from the ionized enol intermediates formed within the interconversion 1^+.?2^+. of the diketone ions but rather from the n-propylidenephthalide ions 4^+.. This follows from CID-MIKE spectrometry of the [M ? CH₃]⁺ ions of 1–4 and two reference C₁₀H₇O₂⁺ (m/z 159) ions of authentic structures (protonated 2-methylene-1,3-indandione and protonated 1,4-naphthoquinone). The characteristic CID fragmentation of the C₁₀H₇O₂⁺ ions is rationalized. Finally, the multistep isomerization of ionized 1,3-indandiones apparently also extends to higher homologues [e.g. 5^+. from 2-ethyl-2-methyl-1,3-indandione (5) and 6^+. from 2,2-diethyl-1,3-indandione (6)]: the ionized phthaloyl group of 1,3-indandione radical cations 1^+., 2^+., 5^+. and 6^+., originally attached with its two acyl functionalities to the same carbon of the aliphatic chain, performs, in fact, a ‘multi-step migration’.